This invention relates to a method of measuring a reactant consumed in a biological reaction. More particular, it relates to a quantitative method for the measurement of the consumption of a substrate in a microbially catalysed reaction. Preferably, it relates to the measurement of biochemical oxygen demand (BOD) of a sample by determining the equivalent mediator demand or alternatively the measurement of the biological toxicity of a sample by the perturbation in quantity of mediator conversion induced by the presence of the sample.
Biochemical oxygen demand is the amount of oxygen taken up owing to the respiratory activity of microorganisms growing on organic compounds present in a water sample when incubated at 20xc2x0 C. for a fixed period (usually five days). It is a measure of the degree of organic pollution of water.
A disadvantage in the conventional method of measuring BOD is that it can take up to five days to do so partly because of the low level of solubility of oxygen in water and partly because of the low microbial concentration in the seed material.
Non-biological methods for rapidly determining the BOD of a sample are described in U.S. Pat. Nos. 3,725,236 and 3,857,761. These patents relate to substantially exhaustive electrochemical oxidation of an aqueous sample in an apparatus for forming oxygen which is measured to calculate BOD.
U.S. Pat. No. 5,085,757 describes another apparatus for rapidly determining BOD of a liquid at an on-site location. The apparatus is a very elaborate one. The method of determining BOD involves a biochemical reaction using a culture medium and measuring the change in the dissolved oxygen content over a period of time and calibrating to determine BOD.
U.S. Pat. No. 5,518,893 is directed to a rapid method of determining BOD. A known microorganism culture is aerated to exhaust the organic matter available to the microorganisms. A sample of liquid to be tested is added and the amount of oxygen consumed by the microorganisms is determined and from this the BOD is calculated. An apparatus for conducting the measurement is described.
GB 2,189,605 describe method for detecting pollution in a continuous liquid flow. The method involves taking a portion of the flowing liquid and adding it to a compatible electron transfer mediator. The mixture is fed into a sensor cell containing bacteria. An activity of the bacteria is stimulated and the level of activity at an electrode in the cell is measured by electron transfer from the mediator.
EP 255291 and EP 470649 both describe methods and an apparatus for making electrochemical measurements or detection of a component of an aqueous liquid sample. The sample containing the component to be analysed is allowed to produce a corresponding quantity of an electrochemically oxidisable or reducible substance. This is then electrochemically oxidised or reduced and the quantity of oxidisable or reduceable substance in the cell is measured as an index of the quantity of the substance.
U.S. Pat. No. 5,413,690 relates to a potentiometric biosensor test strip and a method for detection or measurement of an analyte from a fluid sample. This is done by measuring the change in potential of the system as a result of the chemical reaction of analyte, enzyme and mediator.
It is an object of one aspect of this invention to provide alternatives to the described apparatus and processes or at least to offer the public a useful choice.
Accordingly, the invention may be said broadly to consist of a method of measuring the consumption of a sample substrate in a microbially catalysed biochemical reaction which comprises incubating said substrate with an excess of a microorganism and an excess of a co-substrate until said substrate is oxidised to a predetermined end point and measuring the quantity of reduction of said co-substrate.
In one embodiment the method is used to determine the BOD of said sample substrate.
In another embodiment the method is used to determine the biological toxicity of said sample substrate.
Preferably said reaction is conducted under anaerobic conditions.
In one embodiment said predetermined end point is reached when the rate of change in the reduced co-substrate to oxidised substrate ratio is no longer significant.
In another embodiment said sample substrate and said co-substrate are separated from said microorganism before measuring the quantity of reduction of said co-substrate.
In one embodiment said quantity of reduction of said co-substrate is measured by an electrochemical method.
Alternatively, said quantity of reduction of said co-substrate is measured by an optical method.
Preferably, said electrochemical method is either bulk electrolysis or potentiometry.
Preferably, said optical method is either colorimetry or fluorometry.
In another alternative said quantity of reduction of said co-substrate is measured by reoxidising said mediator and measuring the charge for doing so.
Preferably, said sample substrate contains an organic pollutant.
Alternatively, said sample substrate contains a nutrient pollutant.
Preferably, said micro-organism is any one of Escherichia coli, Proteus vulgaris, Torulopsis candida, Bacillus subtilis, Trichosporon cutaneum and Saccharomyces cerevisiae. 
In one alternative, said microorganism is E. coli. 
In another alternative said microorganism is P. vulgaris. 
Preferably, said co-substrate is any one of benzoquinone, dichlorophenolindophenol, methylene green, methylene blue, phenazine methosulphate, potassium hexacyanoferrate (III), resorufin, thionine, toluidene-blue-O.
Preferably, said anaerobic conditions are maintained by sparging the incubation container in which said incubation is carried out with oxygen free nitrogen.
Preferably, said reoxidation of co-substrate and measurement of charge are carried out in a bulk electrolysis cell.
Preferably, the measured charge is converted to a standard BOD measurement.
Preferably, when necessary said incubation is terminated by filtering said co-substrate, sample substrate and microorganism through a filter whose pore size is such that the co-substrate substrate is in the filtrate and substantially all of the microorganism is in the retentate.
Preferably, the pore size of said filter is 0.45 xcexcm.
Preferably, said co-substrate is a mediator.
The invention may also be said broadly to consist in a method of measuring biochemical oxygen demand substantially as herein described with reference to the examples and FIG. 1.
This invention may also be said broadly to consist in the parts, elements and features referred to or indicated in the specification of the application, individually or collectively, and any or all combinations of any two or more of said parts, elements or features, and where specific integers are mentioned herein which have known equivalents in the art to which this invention relates, such known equivalents are deemed to be incorporated herein as if individually set forth.
The invention consists in the foregoing and also envisages constructions of which the following gives examples.